Shigella dysenteriae Type 1 is one of the most fearsome of the Category B Priority agents owing to its high infectivity at low doses, severe disease sequelae, and ability to cause explosive epidemics in naive populations. No prophylactic vaccine or specific therapy exists for prevention of disease caused by this pathogen and therapeutic options are limited against the majority of clinical isolates which are increasingly multiply antibiotic resistant. S. dysenteriae Type 1 is easily spread by tainted food or water and an intentional act of contamination could lead to widespread serious disease and death in a population with no immunity to this organism. Recent clinical trials with attenuated strains of Shigella flexneri have reinforced the safety and efficacy, of live Shigella vaccine candidates and confirmed the attenuating capacity of mutations in the guaBA, set, and sen genes. These data provide a strategy for the rational design of a series of live, attenuated S. dysenteriae vaccine strains based on a combination of these mutations in addition to elimination of shiga toxin activity. In addition, by inserting a constitutive promoter driving expression of the shiga toxin B subunit in these attenuated strains, shiga toxin neutralizing immune responses could be elicited and provide protection not only against S. dysenterae but also against other shiga toxin-producing pathogens. Using our collection of S. flexneri vaccine strains with known clinical outcomes we propose to modify animal and in vitro assays to increase the sensitivity for distinguishing differences between strains at the preclinical level. Refined assays will allow greater predictive power of subsequent clinical performance resulting in accelerated advancement of the most promising candidate strain or strains to clinical trials and production. The live attenuated strains can then be used to express protective antigens or epitopes from other priority pathogens. We will design systems for expression of PA from B. anthracis from stabilized plasmid constructs, chromosomally integrated and in secreted form by fusion to ClyA. Relevance: These studies will result in a series of live attenuated Shigella dysenteriae vaccine candidates for advancement to clinical trials and for use in the development of a multivalent, oral vaccine to protect against multiple priority agent diseases which will help protect citizens from potential biothreats.